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Purification and demonstration of purity were the primary reasons that techniques were developed for the crystallization of naturally occurring proteins in the laboratory. Protein crystallization was marked by major successes throughout the s and s, with the crystallization of insulin Abel et al. In the s Northrop and coworkers purified a number of important enzymes by crystallization, most notably from the pancreas of pigs and cows reviewed in Northrop et al.
A cascade of successes with other enzymes quickly followed, leading to the award of Nobel Prizes to Sumner and Northrop. The early work of Bernal, Fankuchen, Crowfoot and Perutz Dickerson, made protein crystals important for the three-dimensional structural information that they could potentially yield. The demand for protein crystals expanded rapidly in the s and s as protein crystallography came of age and highly motivated young scientists entered the field.
For 15 years, from about until , X-ray crystallographers depended very much on the successes of earlier protein chemists, and on their somewhat limited procedures and technologies, to provide suitable samples for diffraction.
Ultimately, however, those sources diminished and the methodologies became inadequate. As a result, the s and s saw a great interest develop in devising new approaches to protein crystallization and in discovering and applying new ways to obtain purified samples of novel and biologically important proteins for crystallization McPherson, This endeavor received its greatest boost from an unexpected source: genetics.
With the explosion in genetic engineering and molecular biological research in the s and s came an attendant flood of biologically interesting proteins previously unobtainable because of their low abundance in natural systems. The two disciplines working in tandem, and in many cases tightly coupled, have spawned the structural genomics enterprise, and ultimately promises to allow the detailed visualization of all biological structures at atomic resolution.
This article contains a brief review of the methods and procedures that have emerged from the last years of protein crystal-growth experience. It contains descriptions of the techniques in common use today. It should, however, not be considered to be entirely comprehensive or exhaustive. In particular, it should in no way set boundaries on the imagination and ingenuity of the reader.
There are undoubtedly many contributions yet to be made to this still young, still largely empirical field. Presently, and in the foreseeable future, the only techniques that can yield atomic level structural images of biological macromolecules are X-ray and neutron diffraction as applied to single crystals.
While other methods may produce important structural and dynamic data, for highly precise atomic coordinates only X-ray crystallography is adequate. As its name suggests, application of X-ray crystallography is absolutely dependent on crystals of the macromolecule, and not simply crystals but crystals of sufficient size and quality to permit the collection of accurate diffraction intensities.
The quality of the final structural image is directly determined by the quality of diffraction, that is, the size and physical properties of the crystalline specimen; hence, the crystal becomes the linchpin of the entire process and the ultimate determinant of its success McPherson, General approach Macromolecular crystallization, which includes the crystallization of proteins, nucleic acids and larger macromolecular assemblies such as viruses and ribosomes, is based on a rather diverse set of principles, experiences and ideas.
There is no comprehensive theory, or even a very good base of fundamental data, to guide our efforts, although they are being accumulated at this time. As a consequence, macromolecular crystal growth is largely empirical in nature, and demands patience, perseverance and intuition.
Complicating the entire process, in addition to our limited understanding of the phenomena involved, is the astonishing complexity and range of the macromolecules before us. Even in the case of rather small proteins, such as cytochrome c or myoglobin for example, there are roughly a thousand atoms with thousands of bonds and thousands of degrees of freedom.
For viruses or enzyme complexes having molecular weights measured in the millions of daltons, the possibilities for conformation, interaction and mobility are almost uncountable. Only now are we beginning to develop rational approaches to macromolecular crystallization based on an understanding of the fundamental properties of the systems. We are only now using, in a serious and systematic manner, the classical methods of physical chemistry to determine the characteristics of those mechanisms responsible for the self-organization of large biological molecules into crystal lattices.
As an alternative to the precise and reasoned strategies that we commonly apply to scientific problems, we continue to rely, for the time being at least, on what is fundamentally a trial-and-error approach. Macromolecular crystallization is generally a matter of searching, as systematically as possible, the ranges of the individual parameters that influence crystal formation, finding a set, or multiple sets of factors that yield some kind of crystals, and then optimizing the individual variables to obtain the best possible crystals.
This is usually achieved by carrying out an extensive series, or establishing a vast matrix, of crystallization trials, evaluating the results and using the information that is obtained to improve conditions in successive rounds of trials. Because the number of variables is so large, and because the ranges are so broad, experience and insight in designing and evaluating the individual and collective trials becomes an important consideration.
The nature of protein crystals X-ray analysis is a singular event confined to the research laboratory and the final product is basic scientific knowledge. The crystals themselves, with some exceptions, have no medicinal or pharmaceutical value, but simply serve as intermediaries in the crystallographic process.
The crystals provide the X-ray diffraction patterns that in turn serve as the raw data which allow the direct visualization of the macromolecules or their complexes that the crystals are composed of. Figure 1 Microphotographs of protein and virus crystals grown in the laboratory of AM showing the variety of habits common to macromolecular crystals.
When crystallizing proteins for X-ray diffraction analysis, one is usually dealing with homogenous, often exceptionally pure macromolecules, and the objective may be to grow only a few large, high-quality, high-performance crystals. It is important to emphasize that while the number of crystals needed may be few, often the amount of protein available may be severely limited.
This in turn places grave constraints on the approaches and strategies that may be used to obtain those crystals. Protein or nucleic acid occupies the remaining volume so that the entire crystal is in many ways an ordered gel permeated by extensive interstitial spaces through which solvent and other small molecules freely diffuse.
In proportion to its molecular mass, the number of bonds salt bridges, hydrogen bonds, hydrophobic interactions that a conventional molecule forms to its neighbors in a crystal far exceeds the very few exhibited by crystalline macromolecules. Living systems are based almost exclusively on aqueous chemistry within narrow ranges of temperature and pH. Macromolecules have thus evolved an appropriate compatibility, and serious deviations or perturbations are rarely tolerated.
As a consequence, all protein and nucleic acid crystals must be grown from aqueous solutions to which they are tolerant, and these solutions are called mother liquors.
Macromolecular crystals have so far only been grown from such media. Although comparable in their morphologies and appearance, there are important practical differences between crystals of low-molecular-mass compounds and crystals of proteins and nucleic acids. Crystals of conventional molecules are characterized by firm lattice forces, are relatively highly ordered, are generally physically hard and brittle, are easy to manipulate, can usually be exposed to air, have strong optical properties and diffract X-rays intensely.
Macromolecular crystals are, by comparison, usually more limited in size, are very soft and crush easily, disintegrate if allowed to dehydrate, exhibit weak optical properties and diffract X-rays poorly. Macromolecular crystals are temperature sensitive and undergo extensive damage after prolonged exposure to radiation. Frequently, several or even many crystals must be analyzed for a structure determination to be successful, although the advent of cryocrystallography Pflugrath, , CCD area detectors of very high photon-counting efficiency and dynamic range Gruner et al.
The extent of the diffraction pattern from a crystal is directly correlated with its degree of internal order. The more vast the pattern, or the higher the resolution to which it extends, the more structurally uniform are the molecules in the crystal and the more precise is their periodic arrangement.
The level of detail to which atomic positions can be determined by crystal structure analysis corresponds closely with this degree of crystalline order.
While conventional crystals often diffract to their theoretical limit of resolution, protein crystals, by comparison, produce diffraction patterns of more limited extent.
The liquid channels and solvent-filled cavities that permeate macromolecular crystals are primarily responsible for the limited resolution of the diffraction patterns. Because of the relatively large spaces between adjacent molecules and the consequent weak lattice forces, all molecules in the crystal may not occupy exactly equivalent orientations and positions, but may vary slightly within or between unit cells.
Furthermore, because of their structural complexity and their potential for conformational dynamics, protein molecules in a particular crystal may exhibit slight variations in the course of their polypeptide chains or in the dispositions of side groups from one to another. Although the presence of extensive solvent regions is a major contributor to the generally modest diffraction quality of protein crystals, it is also responsible for their value to biochemists.
Because of the high solvent content, the individual macromolecules in protein crystals are surrounded by layers of water that maintain their structure virtually unchanged from that found in solution. As a consequence, ligand binding, enzymatic, spectroscopic characteristics and most other biochemical features are essentially the same as for the fully solvated molecule. Conventional chemical compounds, which may be ions, ligands, substrates, coenzymes, inhibitors, drugs or other effector molecules, may be freely diffused into and out of the crystals.
Crystalline enzymes, although immobilized, are completely accessible for experimentation simply through alteration of the surrounding mother liquor. Polymorphism, as is evident in Fig. Presumably this is a consequence of their conformational dynamic range and the sensitivity of the lattice contacts involved.
Thus, different habits and different unit cells may arise from what, by most standards, would be called identical conditions. In fact, multiple crystal forms are sometimes seen coexisting in the same sample of mother liquor. There are further differences which complicate the crystallization of macromolecules compared with conventional small molecules Feigelson, ; Feher,
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Purification and demonstration of purity were the primary reasons that techniques were developed for the crystallization of naturally occurring proteins in the laboratory. Protein crystallization was marked by major successes throughout the s and s, with the crystallization of insulin Abel et al. In the s Northrop and coworkers purified a number of important enzymes by crystallization, most notably from the pancreas of pigs and cows reviewed in Northrop et al. A cascade of successes with other enzymes quickly followed, leading to the award of Nobel Prizes to Sumner and Northrop. The early work of Bernal, Fankuchen, Crowfoot and Perutz Dickerson, made protein crystals important for the three-dimensional structural information that they could potentially yield.
Recent advances in the microgravity crystallization of biological macromolecules.
McPherson and B. Cudney For the successful X-ray structure determination of macromolecules, it is first necessary to identify, usually by matrix screening, conditions that yield some sort of crystals. Initial crystals are frequently microcrystals or clusters, and often have unfavorable morphologies or yield poor diffraction intensities. It is therefore generally necessary to improve upon these initial conditions in order to obtain better crystals of sufficient quality for X-ray data collection.
Crystallization of biological macromolecules
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